Reverse transcription is a critical step in the life cycle of all RNA tumor viruses, also known as retroviruses because the retroviruses integrate their DNA into the host cell DNA by a reverse transcriptase (RT), also known as an RNA-dependent DNA polymerase, wherein the RT directs the synthesis of a complementary DNA (cDNA) from an RNA template.
An RT is a DNA polymerase enzyme that is encoded by retroviruses, which use the enzyme during the process of replication. Reverse-transcribing RNA viruses, such as retroviruses, use the enzyme to reverse-transcribe their RNA genomes into DNA, which is then integrated into the host genome and replicated along with it. The virus thereafter replicates as part of the host cell's DNA. Without reverse transcriptase, the viral genome would not be able to incorporate into the host cell, resulting in the failure of the ability to replicate.
Both viral and cloned RTs contain two enzymatic activities: DNA polymerase activity and ribonuclease H (RNase H) activity. In the retrovirus life cycle, DNA polymerase activity is responsible for transcribing viral RNA into double-stranded DNA. RNase H activity, on the other hand, degrades RNA from RNA-DNA hybrids, such as are formed during reverse transcription of an RNA template.
Retroviral RNase H, a part of the viral reverse transcriptase enzyme, is an important pharmaceutical target, as it is absolutely necessary for the proliferation of retroviruses, such as HIV and murine leukemia virus (M-MLV). Mizuno, M., Yasukawa K, Inouye K. Insight into the Mechanism of the Stabilization of Moloney Murine Leukemia Virus Reverse Transcriptase by Eliminating RNase H activity. Biosci. Biotechnol. Biochem. 74 (2):440-2 (2010); Coté M L, Roth M J. Murine leukemia virus reverse transcriptase: structural comparison with HIV-1 reverse transcriptase. Virus Res. 134 (1-2): 186-202 (2008).
As a result, RT is used extensively in recombinant DNA technology to synthesize cDNA from mRNA. One major problem with cDNA synthesis is that the RNase H activity of RT degrades the mRNA template during first-strand synthesis. The mRNA poly(A)-oligo(dT) hybrid used as a primer for first-strand cDNA synthesis is degraded by RT RNase H. Thus, at the outset of cDNA synthesis, a competition is established between RNase H-mediated deadnylation of mRNA and initiation of DNA synthesis, which reduces the yield of cDNA product, Berger, S. L., et al., Biochem. 22:2365-73 (1983), and often causes premature termination of DNA chain growth.
Accordingly, a need for developing conditions, which are more efficient for supporting cDNA synthesis, sequencing, and amplification in reverse transcription, has been present for a long time. This invention is directed to solve these problems and satisfy a long-felt need.